Dendrobium Nobile Alcohol Extract Extends the Lifespan of Caenorhabditis elegans via hsf-1 and daf-16

Dendrobium nobile is a traditional Chinese herb with anti-inflammatory, antioxidant, and neuroprotective properties. However, its antiaging effects are unclear. Herein, we studied the aging-related functions and the mechanism of action of the alcohol extract of Dendrobium nobile (DnAE) in the model organism Caenorhabditis elegans. The results indicated that 1 mg/mL DnAE slowed lipofuscin accumulation, decreased the levels of reactive oxygen species, elevated superoxide dismutase activity, enhanced oxidative and heat stress resistance, extended the lifespan of nematodes, protected their dopamine neurons from 6-hydroxydopamine-induced neurodegeneration, and reduced Aβ-induced neurotoxicity. DnAE upregulated the mRNA expression of the transcription factors DAF-16 and HSF-1, promoted the nuclear localization of DAF-16, and enhanced the fluorescence intensity of HSP-16.2. However, it had no effect on the lifespan of DAF-16 mutants. Thus, DnAE can significantly extend lifespan, enhance heat stress tolerance, and delay age-related diseases through a DAF-16-dependent pathway.


Introduction
Aging is a natural process characterized by structural deterioration, loss of bodily functions, and decreased immunity [1].Various studies have shown that aging is a significant risk factor for degenerative diseases, including cardiovascular [2] and neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD) [3].Natural active ingredients are widely used in antiaging research due to their versatile structures and multitarget binding.For instance, resveratrol is a recognized natural active ingredient in the antiaging process that can extend the lifespan of mice through caloric restriction [4].Blueberry extract has been shown to promote the lifespan and stress tolerance of Caenorhabditis elegans (C.elegans) via DAF-16 [5], and ginsenosides extend the lifespan of C. elegans and reduce the accumulation of lipofuscin [6], a complex of oxidized lipids and proteins that accumulates during cellular and tissue senescence that is often considered a marker of senescence in C. elegans [7].Given the increasing global aging problem, developing natural products that can delay aging or promote a healthy lifespan is crucial.
Dendrobium nobile Lindl is an epiphytic herb of the genus Dendrobium from the family Orchidaceae.It is traditionally used in Chinese medicine to nourish yin, clear heat, and promote overall health [8].Modern research has highlighted D. nobile's significant biological effects in reducing cancer risk [9], preventing diabetes [10], improving oxidative stress [11], and preventing neurodegenerative diseases such as AD and PD [12].It mainly consists of alkaloids, polysaccharides, and phenolic compounds.Studies have demonstrated that D. nobile alkaloids exert beneficial effects on glucose and lipid metabolism in mouse livers [13], attenuate LPS-induced tau protein hyperphosphorylation in rat hippocampi, and prevent LPS-induced apoptosis in rat brains [14].Its phenolic compounds exhibit anti-inflammatory activity by targeting various inflammation-associated cytokines [15].Synergistic interactions between multiple compounds from the same plant can provide enhanced biological activity [16].Our previous results showed that the alcohol extract of D. nobile (DnAE) could exert immune-enhancing, antioxidant, and antiaging effects by remodeling the intestinal microecosystem and downregulating the expression of proaging genes in mouse brains [17].Therefore, we obtained the total extract of D. nobile using alcohol as the most suitable solvent and investigated whether the extract could delay aging and prevent age-related diseases.
C. elegans is a popular model for studying stress resistance and longevity due to its short lifespan, mature genetic pathways, and high reproductive rate [18].Stress resistance and aging in C. elegans are regulated by multiple signaling pathways including the insulin/insulin-like growth factor (IIS) pathway, mitogen-activated protein kinase pathway, diet restriction pathway, germline pathway, and key transcription factors such as SKN-1 [19].The IIS is highly evolutionarily conserved and is the first pathway found to significantly affect the longevity of various species.Reduced IIS increases cellular tolerance to a variety of stresses, thus prolonging lifespan [20].DAF-16 and HSF-1 are the two key transcription factors of the IIS pathway in C. elegans.DAF-16 is the most extensively studied factor to prolong the lifespan of C. elegans, one important mechanism of which is that DAF-16 reduces reactive oxygen species (ROS) levels and increases the activities of antioxidant enzymes such as superoxide dismutase (SOD), glutathione S-transferase (GST), and catalase by regulating the downstream genes sod-3, gst-4, and ctl-1 [21][22][23].HSF-1 is involved in modulating the lifespan and longevity pathways of C. elegans by regulating the expression of various stress proteins such as heat shock proteins (HSPs) [24].
In the present study, we investigated the antiaging performance of DnAE and its molecular mechanism by detecting the vitality and antioxidant biomarkers of C. elegans.Furthermore, we identified that DnAE is neuroprotective and delays nematode aging by activating the stress-related transcription factors DAF16/FOXO and HSF-1 from the IIS pathway.These findings provide new insights into the potential protective role of D. nobile in healthy aging and aging-related diseases.

Chemical Composition of DnAE
The composition characteristics of DnAE were determined by UHPLC-Q-TOF-MS/MS in both positive and negative ion modes (Figure 1).The compounds were initially identified by comparisons using the Shanghai Applied Protein Technology database and public databases Mass Bank, Metlin, HMDB, and MoNA and subsequently confirmed by standards and the literature (Figure S1).As shown in Table 1, DnAE was mainly composed of 27 known constituents, which accounted for 95% of the total peaks observed and included palmitic amide, glyceryl monostearate, linoleic acid, mubironine B, quinic acid, dendroside G, and dendrobine.Eight components were qualified by the standards, and the remaining components were tentatively identified based on the library and/or literature data.

DnAE Increases the Lifespan and Improves the Health Span of C. elegans
To investigate the effects of DnAE on the lifespan of C. elegans and determine the optimal dosage, we treated C. elegans with different DnAE concentrations.DnAE significantly extended lifespan, with the best effect observed at a concentration of 1 mg/mL, which increased the lifespan by up to 21.37% (p = 0.0013) (Figure 2A and Table S1).A control experiment was conducted using an equivalent alcohol concentration to exclude the effects of solvents on the experimental results.The lifespan of C. elegans was not significantly different between ddH 2 O treatment and alcohol treatment (Figure 2B and Table S2).The reproductive toxicity of a drug can be reflected by the number of eggs produced by the worm [32].A comparable number of eggs were produced by the treatment and control groups (Figure 2C and Table S3), indicating the reproductive safety of DnAE.Dietary restriction and muscle activity can also affect the lifespan of worms [33].Supplementation with DnAE caused an imperceptible change in the number of bends and swallows of C. elegans within a certain timeframe (Figure 2D,E and Tables S4 and S5).The level of lipofuscin is a commonly used indicator of aging in C. elegans [34].We used the spontaneous fluorescence intensity of lipofuscin to measure its content in the worms.The results revealed increasing lipofuscin levels with increasing age (Figure 2F and Table S6).We measured the fluorescence levels of C. elegans treated with or without DnAE treatment on the 5th and 10th days and found that 1 mg/mL DnAE significantly reduced the increase in lipofuscin.The difference was most significant on the 5th day but decreased over time, consistent with the results showing an increase in the lifespan of C. elegans (Figure 2F).

DnAE Increased Heat Stress Tolerance by Upregulating the Expression of hsf-1 and HSPs
Due to the close relationship between lifespan extension and stress resistance [24], we further investigated whether DnAE could extend the lifespan of C. elegans under thermal stress.In the thermal stress experiment, wild-type worms were cultured for 5 days and then exposed to 37 °C.Their survival and mortality rates were recorded hourly.The results showed that, compared to the control group, DnAE increased the survival rate of C. elegans under heat stress and extended their lifespan by 34.09% (p = 0.007) (Figure 3A and Table S7).HSF-1 is the main transcription factor that affects the heat resistance of C. elegans, and downstream target proteins such as HSP-16.2,HSP-6, and HSP-60 are involved in the response to stress.In the present study, DnAE significantly increased the mRNA levels of hsf-1-and hsp-related genes (Figure 3B and Table S14).To confirm the link between HSF-1 and DnAE, we quantified hsp-16.2expression using the hsp-16.2p::greenfluorescent protein (GFP) mutant strain TJ375.Our results showed that DnAE enhanced the fluorescence level of TJ375 cells (Figure 3C and Table S10), while it lost the ability to extend the nematode lifespan in the hsf-1 ineffective mutant hsf-1(sy441 and Table S11) (Figure 3D).

DnAE Increased Heat Stress Tolerance by Upregulating the Expression of hsf-1 and HSPs
Due to the close relationship between lifespan extension and stress resistance [24], we further investigated whether DnAE could extend the lifespan of C. elegans under thermal stress.In the thermal stress experiment, wild-type worms were cultured for 5 days and then exposed to 37 • C. Their survival and mortality rates were recorded hourly.The results showed that, compared to the control group, DnAE increased the survival rate of C. elegans under heat stress and extended their lifespan by 34.09% (p = 0.007) (Figure 3A and Table S7).HSF-1 is the main transcription factor that affects the heat resistance of C. elegans, and downstream target proteins such as HSP-16.2,HSP-6, and HSP-60 are involved in the response to stress.In the present study, DnAE significantly increased the mRNA levels of hsf-1and hsp-related genes (Figure 3B and Table S14).To confirm the link between HSF-1 and DnAE, we quantified hsp-16.2expression using the hsp-16.2p::greenfluorescent protein (GFP) mutant strain TJ375.Our results showed that DnAE enhanced the fluorescence level of TJ375 cells (Figure 3C and Table S10), while it lost the ability to extend the nematode lifespan in the hsf-1 ineffective mutant hsf-1(sy441 and Table S11) (Figure 3D).

DnAE Enhances Antioxidant Activity in C. elegans
Oxidative damage is a key factor in aging and manifests as the level of free radicals in the body [35].Consequently, drugs with antioxidant capacity are often considered almost analogous to antiaging drugs.To investigate the effect of DnAE on the antioxidant capacity of C. elegans, we exposed the nematodes to hydrogen peroxide-and paraquatinduced oxidative environments and measured their survival time.The DnAE-treated groups exhibited excellent antioxidant capacity, with average lifespan extensions of 23.16% and 21.87% under acute and chronic oxidative stress, respectively, compared to those in the control group (p < 0.001) (Figure 4A,B and Table S7).Additionally, we measured the following molecular markers of antioxidant defense: SOD and ROS.ROS are oxidative stress substances produced within cells, and SOD can help clear ROS and reduce the degree of oxidative stress reactions.DnAE prolonged the lifetime of N2 under oxidative stress, reduced ROS levels, and increased SOD activity (Figure 4C,D and Tables S8 and S9).

DnAE Promotes the Nuclear Localization of DAF-16 and the Expression of SOD-3 an GST-4
To gain a deeper understanding of the molecular mechanism by which DnA tends the lifespan of wild-type C. elegans in view of the beneficial effects of DnAE o and oxidative stress, we tested whether DnAE could affect the transcriptional acti the previously studied longevity-associated transcription factor DAF-16.DAF-16 ortholog of the forkhead box O (FOXO) family in humans [36].The activation of D can lead to the increased expression of antioxidant enzymes, such as SOD, which protect cells against oxidative damage and contributes to lifespan extension.We us

DnAE Promotes the Nuclear Localization of DAF-16 and the Expression of SOD-3 and GST-4
To gain a deeper understanding of the molecular mechanism by which DnAE extends the lifespan of wild-type C. elegans in view of the beneficial effects of DnAE on heat and oxidative stress, we tested whether DnAE could affect the transcriptional activity of the previously studied longevity-associated transcription factor DAF-16.DAF-16 is the or-tholog of the forkhead box O (FOXO) family in humans [36].The activation of DAF-16 can lead to the increased expression of antioxidant enzymes, such as SOD, which helps protect cells against oxidative damage and contributes to lifespan extension.We used the TJ356 daf-16p::daf-16a/b::GFP mutant to study the subcellular localization of DAF-16 and the expression of its target genes.As shown in Figure 5A, DnAE increased the number of worms with nuclear localization of DAF-16 after treatment with paraquat.As SOD-3 and GST-4 are essential downstream target proteins of the DAF-16 transcription factor, their expression was visualized by exploiting the CF1553 and CL2166 mutant strains (Figure 5B,C).Compared to that in the control group, the fluorescence intensity in the DnAE treatment group was significantly enhanced, implying increased SOD-3 and GST-4 expression.Paraquat substantially depletes antioxidant enzymes in C. elegans.DnAE increased the fluorescence intensity of SOD-3 and GST-4 after treatment with 4 mM paraquat, enhancing C. elegans resistance (Table S10).

DnAE-Mediated Lifespan Extension in C. elegans Is Dependent on DAF-16
Apart from DAF-16, the transcription factor SKN-1 is another well-known lon factor that plays an important role in the oxidative stress response.To clarify whi naling pathway is regulated by DnAE to prolong the lifespan of C. elegans, we exa the expression of skn-1, daf-16, and related target genes in N2 under DnAE treatmen findings demonstrate that while skn-1 expression remained unaffected, DnAE cantly upregulated the expression of daf-16 and its downstream genes such as sod-3 and ctl-1 and decreased the expression of its upstream gene daf-2 (Figure 6A and S14).Furthermore, after DnAE administration, we found that DnAE had no effect lifespan of daf-16 (mu86) or daf-2 (e1370) mutant strains (Figure 6B,C and Table S11 encodes the sole C. elegans homolog of the insulin/IGF-1 receptor, and its degra stimulates DAF-16 activity, promoting longevity [37].Therefore, the extension lifespan of C. elegans by DnAE mainly depends on DAF-16 from the IIS pathway.

DnAE-Mediated Lifespan Extension in C. elegans Is Dependent on DAF-16
Apart from DAF-16, the transcription factor SKN-1 is another well-known longevity factor that plays an important role in the oxidative stress response.To clarify which signaling pathway is regulated by DnAE to prolong the lifespan of C. elegans, we examined the expression of skn-1, daf-16, and related target genes in N2 under DnAE treatment.Our findings demonstrate that while skn-1 expression remained unaffected, DnAE significantly upregulated the expression of daf-16 and its downstream genes such as sod-3, gst-4, and ctl-1 and decreased the expression of its upstream gene daf-2 (Figure 6A and Table S14).Furthermore, after DnAE administration, we found that DnAE had no effect on the lifespan of daf-16 (mu86) or daf-2 (e1370) mutant strains (Figure 6B,C and Table S11).daf-2 encodes the sole C. elegans homolog of the insulin/IGF-1 receptor, and its degradation stimulates DAF-16 activity, promoting longevity [37].Therefore, the extension of the lifespan of C. elegans by DnAE mainly depends on DAF-16 from the IIS pathway.

DnAE Decreases the Progression of Aging-Related Diseases
PD is characterized by the accumulation of α-synuclein in the substantia nigra and the degeneration of dopaminergic neurons [38].C. elegans lacks a homolog of α-synuclein, so a transgenic strain expressing human α-synuclein in body wall muscle cells was created to study the pathogenicity of α-synuclein [39].The transgenic strain NL5901 expresses αsynuclein fused with yellow fluorescent protein (YFP).Dietary restriction (DR) significantly inhibits protein toxicity and age-related paralysis in the C. elegans model through a mechanism distinct from the IIS pathway [40].After treatment with 1 mg/mL DnAE, we observed no noticeable change in α-synuclein aggregation on the first day, but there was a significant reduction after five days.Additionally, DnAE treatment notably enhanced the inhibition of α-synuclein aggregation compared to that in the positive control group under DR conditions (Figure 7A and Table S12).Dopaminergic neuron degeneration is readily induced by neurotoxins such as 6-hydroxydopamine (6-OHDA) [41].Therefore, another transgenic strain, BZ555, expressing GFP in dopaminergic neurons, was used to study dopaminergic neuron degeneration.After treating C. elegans with 6-OHDA for 72 h, fluorescence images of its head neurons were taken.The results demonstrated that 6-OHDA treatment reduced the average fluorescence intensity of BZ555 in the absence of DnAE, while the addition of DnAE improved this effect (Figure 7B).These results revealed DnAE-delayed dopaminergic neuron degeneration in BZ555.Its protective effect was equivalent to that of the anti-PD drug levodopa (Figure 7B and Table S12).
β-Amyloid is the main cause of neuronal degeneration and death around senile plaques in the brains of AD patients.High temperatures can induce its expression in the muscle of the transgenic strain CL4176, resulting in paralysis of the strain [42].DnAE delayed the paralysis of the CL4176 strain and reduced the toxicity of the β-amyloid protein, In the DR model, CL4176 paralysis was delayed by 7.01%, which increased to 10.01% in the presence of DnAE (Figure 7C and Table S13).

DnAE Decreases the Progression of Aging-Related Diseases
PD is characterized by the accumulation of α-synuclein in the substantia nigra and the degeneration of dopaminergic neurons [38].C. elegans lacks a homolog of α-synuclein, so a transgenic strain expressing human α-synuclein in body wall muscle cells was created to study the pathogenicity of α-synuclein [39].The transgenic strain NL5901 expresses α-synuclein fused with yellow fluorescent protein (YFP).Dietary restriction (DR) significantly inhibits protein toxicity and age-related paralysis in the C. elegans model through a mechanism distinct from the IIS pathway [40].After treatment with 1 mg/mL DnAE, we observed no noticeable change in α-synuclein aggregation on the first day, but there was a significant reduction after five days.Additionally, DnAE treatment notably enhanced the inhibition of α-synuclein aggregation compared to that in the positive control group under DR conditions (Figure 7A and Table S12).Dopaminergic neuron degeneration is readily induced by neurotoxins such as 6hydroxydopamine (6-OHDA) [41].Therefore, another transgenic strain, BZ555, expressing GFP in dopaminergic neurons, was used to study dopaminergic neuron degeneration.After treating C. elegans with 6-OHDA for 72 h, fluorescence images of its head neurons were taken.The results demonstrated that 6-OHDA treatment reduced the average fluorescence intensity of BZ555 in the absence of DnAE, while the addition of DnAE improved this effect (Figure 7B).These results revealed DnAE-delayed dopaminergic neuron degeneration in BZ555.Its protective effect was equivalent to that of the anti-PD drug levodopa (Figure 7B and Table S12).
β-Amyloid is the main cause of neuronal degeneration and death around senile plaques in the brains of AD patients.High temperatures can induce its expression in the muscle of the transgenic strain CL4176, resulting in paralysis of the strain [42].DnAE delayed the paralysis of the CL4176 strain and reduced the toxicity of the β-amyloid protein, In the DR model, CL4176 paralysis was delayed by 7.01%, which increased to 10.01% in the presence of DnAE (Figure 7C and Table S13).

Discussion
Various components of DnEA have significant pharmacological effects.Dendrobine, known for its antiaging and neuroprotective effects, inhibits dopamine-related neuronal apoptosis in PD models [43] and reduces SASP factor expression in chondrocytes, suggesting potential for osteoarthritis treatment [44].Dendroxine protects PC12 cells from Aβ1-42-induced neurotoxicity by inhibiting CDK5 [45].Nobilonine reduces lipopolysaccharides in rats [46].With a wide range of physiological functions, oleic acid reduces the risk of cardiovascular disease, autoimmune disorders, and cancer and aids wound healing [47].Therefore, DnAE has notable pharmacological effects on aging and aging-related diseases.
Contrary to earlier findings [30,48], DnAE increased C. elegans' longevity and slowed the buildup of lipofuscin, but it had no effect on their bending frequency, pharyngeal pumping rate, or ability to lay eggs over a given amount of time.Thus, we suggest that DnAE could function via a different mechanism than calorie restriction, increased exercise, or the suppression of reproduction.
There is a strong correlation between the extension of the lifespan of C. elegans and their ability to resist stress, including heat stress.DnAE significantly increased the survival time of C. elegans exposed to high temperatures, enhancing heat resistance.Additionally, DnAE upregulated the expression of hsf-1 and HSPs.HSF-1 is a transcription factor that is pivotal in the heat stress response.When cells experience adverse environmental conditions such as high temperatures and oxidative stress, HSF-1 can activate the expression of a series of HSPs that protect cells from stress-induced damage.HSPs act as molecular chaperones and regulate protein folding, thereby safeguarding other proteins against stress-induced damage [40].Some HSPs have been

Discussion
Various components of DnEA have significant pharmacological effects.Dendrobine, known for its antiaging and neuroprotective effects, inhibits dopamine-related neuronal apoptosis in PD models [43] and reduces SASP factor expression in chondrocytes, suggesting potential for osteoarthritis treatment [44].Dendroxine protects PC12 cells from Aβ1-42-induced neurotoxicity by inhibiting CDK5 [45].Nobilonine reduces lipopolysaccharides in rats [46].With a wide range of physiological functions, oleic acid reduces the risk of cardiovascular disease, autoimmune disorders, and cancer and aids wound healing [47].Therefore, DnAE has notable pharmacological effects on aging and aging-related diseases.
Contrary to earlier findings [30,48], DnAE increased C. elegans' longevity and slowed the buildup of lipofuscin, but it had no effect on their bending frequency, pharyngeal pumping rate, or ability to lay eggs over a given amount of time.Thus, we suggest that DnAE could function via a different mechanism than calorie restriction, increased exercise, or the suppression of reproduction.
There is a strong correlation between the extension of the lifespan of C. elegans and their ability to resist stress, including heat stress.DnAE significantly increased the survival time of C. elegans exposed to high temperatures, enhancing heat resistance.Additionally, DnAE upregulated the expression of hsf-1 and HSPs.HSF-1 is a transcription factor that is pivotal in the heat stress response.When cells experience adverse environmental conditions such as high temperatures and oxidative stress, HSF-1 can activate the expression of a series of HSPs that protect cells from stress-induced damage.HSPs act as molecular chaperones and regulate protein folding, thereby safeguarding other proteins against stress-induced damage [40].Some HSPs have been shown to inhibit virus proliferation by interacting with viruses and activating the immune pathway to protect host cells during viral infections, indicating antiviral properties [49].These findings are consistent with the reported antiviral effects of D. nobile [50].Therefore, DnAE delayed the aging process of C. elegans by increasing its tolerance to adversity by upregulating the expression of hsf-1 and HSPs.
D. nobile contains several chemical components that exert antioxidant effects [51].Consistent with this finding, DnAE significantly extended the survival time of C. elegans exposed to H 2 O 2 and paraquat.We believe that the antioxidant properties of DnAE are crucial for delaying the aging process.
SKN-1, a regulatory factor for C. elegans' responses to environmental stress, is a homologous gene to Nrf2 [52,53].Despite some evidence that D. nobile activates the Nrf2 pathway to exert its biological activity [11,54,55], we discovered that DnAE did not increase skn-1 expression.Daf-16 activation and Daf-2 degradation are the primary regulators of the IIS pathway that prolong the lifespan of C. elegans [56].According to our findings, DnAE promoted DAF-16 entry into the nucleus through the cytoplasm and enhanced the expression of daf-16 and its downstream regulatory genes sod-3, ctl-1, and gst-4.Moreover, DnAE downregulated the expression of daf-2, which stimulates DAF-16 activity but did not extend the lifespan of the daf-16(mu86) or daf-2(e1370) mutant strains.These findings suggest that DnAE may prolong the lifespan of C. elegans by inhibiting the IIS pathway.
Neurodegenerative diseases caused by aging have always been a challenge in medicine.Water extracts of D. nobile can inhibit neuronal apoptosis, enhance the expression of nerve growth factors, and provide neuroprotection for rats with hypoxic-ischemic brain injury [57].Since nematodes lack homologous genes for PD/AD, we used transgenic nematode strains and found that DnAE protected DA neurons from 6-OHDA-induced neurodegeneration, reduced the aggregation of α-synuclein, and lowered Aβ-induced neurotoxicity.This finding is consistent with the neuroprotective effect of D. nobile extract in other animal models [58].According to previous reports, DR inhibits protein toxicity and age-related paralysis in C. elegans models through pathways unrelated to daf-16 [40].In the DR model, DnAE strengthened the inhibition of α-synuclein aggregation and delayed the paralysis induced by Aβ, indicating that DnAE exerts neuroprotective effects through pathways different from those in DR.In addition, research has shown a close relationship between DAF-16 and the nervous system.For example, gastrodin reduces α-synuclein accumulation in nematodes through the DAF-2/DAF-16 signaling pathway [59], and cannabidivarin prevents DA-related neuronal degeneration through DAF-16 [60].The activation of DAF-16 improves the royal jelly protein balance and reduces β-amyloid toxicity [61].Therefore, we speculated that D. nobile extract provides neuroprotection by promoting DAF-16 expression, but extensive experimental data are required to confirm this.

Conclusions
DnAE slowed the accumulation of lipofuscin, reduced the ROS content, and increased the SOD activity of N2.Moreover, it prolonged the lifespan of N2 under oxidative stress and heat stress, which was achieved by upregulating the expression of daf-16 and hsf-1, promoting the nuclear localization of DAF-16, and increasing the expression of sod-3, gst-4, and ctl-1.Additionally, DnAE alleviated 6-OHDA-induced DA-related neuronal degeneration and reduced Aβ-induced toxicity.These results highlight the potential of DnAE for further investigation in extending lifespan and treating age-related diseases (Figure 8).

Strains and DnAE Preparation
The following strains were obtained from the Caenorhabditis Genetics Center (CGC) and maintained at the appropriate temperature as per previous reports [62] unless otherwise specified: N2 (Bristol, wild-type) [63] (Ziyang, China).It was dried in an oven at 60 °C and pulverized.The resulting powder was sifted through an 80-mesh sieve, mixed with petroleum ether, and subjected to ultrasonic oscillation for 30 min to produce a solid-liquid mixture.The mixture was subsequently filtered, and the remaining residues were ultrasonicated with 80%, 75%, and 70% ethyl alcohol (1:50 g/mL w/v) at 50 °C for 30 min before refiltering.Finally, all alcohol extracts were combined and evaporated to obtain DnAE.

Strains and DnAE Preparation
The following strains were obtained from the Caenorhabditis Genetics Center (CGC) and maintained at the appropriate temperature as per previous reports [62] unless otherwise specified: N2 (Bristol, wild-type) [63]  C and pulverized.The resulting powder was sifted through an 80-mesh sieve, mixed with petroleum ether, and subjected to ultrasonic oscillation for 30 min to produce a solid-liquid mixture.The mixture was subsequently filtered, and the remaining residues were ultrasonicated with 80%, 75%, and 70% ethyl alcohol (1:50 g/mL w/v) at 50 • C for 30 min before refiltering.Finally, all alcohol extracts were combined and evaporated to obtain DnAE.

UHPLC-Q-TOF-MS/MS Compound Identification
Compound identification of DnAE was carried out using a UHPLC Agilent 12900 series instrument (Agilent Technologies, Santa Clara, CA, USA) and an ACQUITY UPLC BEH C-18 (Waters Corp, Milford, MA, USA) analytical column (100 mm × 2.1 mm).An AB Triple TOF 6600 (SCIEX, Framingham, MA, USA) mass spectrometer was used to collect the primary and secondary spectra of the compounds.The chromatographic and mass spectrometry conditions followed previous methods [69].The structures of the compounds were identified by comparing their molecular weights (molecular mass errors less than 10 ppm), secondary fragmentation determination spectra, and retention times in the Shanghai Applied Protein Technology database and public databases Mass Bank, Metlin, HMDB, and MoNA.These results were subsequently checked and confirmed by comparison with the standards and literature.

Lifespan Assay
DnAE was diluted with water to concentrations of 200 µg/mL, 500 µg/mL, and 1 mg/mL after being dissolved in ethanol to a concentration of 80 mg/mL.DnAE was applied to the prepared NGM plates at various concentrations and allowed to blow dry, after which, 200 µL OP50 was added to the NGM plates.At least 60 synchronized L4-stage C. elegans were selected and placed on NGM plates for the treatment and control groups.The time of synchronization was recorded as day 0. The worms were cultured at 20 • C, transferred to new NGM plates with or without drugs every 24 h, and counted for survival, death, or missing numbers.To accurately capture the number of deaths during natural growth, dead worms were counted beginning on day 8. Worms that did not respond when lightly touched on the head with a worm pick were considered dead.The experiment was repeated three times, as shown in Table S1.

Reproduction Assay
Three synchronized L4-stage C. elegans were selected and equally divided into treatment and control groups.The worms were transferred to NGM plates containing 1 mg/mL DnAE for the treatment group or solvent without DnAE for the control group.The worms were transferred to new NGM plates every 24 h, and the number of offspring produced on the previous plates was recorded.Self-fertilization of C. elegans was observed for 5 days after the L4 stage to calculate the average and total number of eggs produced per hermaphroditic worm.The experiment was repeated three times and the numbers of C. elegans per treatment are shown in Table S.

Bending Assay
This study aimed to assess the effect of D. nobile on C. elegans' motility.At least 20 synchronized L4-stage worms were transferred to NGM plates containing 1 mg/mL DnAE or DnAE-free solvent for the treatment or control groups, respectively.The worms were transferred to new NGM plates every 48 h and cultured at 20 • C for 72 h before the number of body bends was recorded for 20 s.These measurements were used to assess motility.The experiment was repeated three times, as shown in Table S5.

Pharyngeal Pumping Assay
At least 20 synchronized L4-stage C. elegans were transferred to NGM plates and treated with 1 mg/mL DnAE or blank solvent for the treatment or control groups, respectively.The treatment group was further divided into subgroups according to DnAE concentrations.The worms were transferred to new NGM plates every 48 h and cultured at 20 • C for 72 h.Then, the worms were observed using a microscope, and the number of pharyngeal pumping events during a 10 s period was recorded.The experiment was repeated three times, as shown in Table S4.

Oxidative Stress Resistance Assay
The dosing and control groups were treated with or without 1 mg/mL DnAE.At least 60 synchronized L4-stage nematodes were selected and transferred to NGM plates for the treatment and control groups.After 48 h, the NGM plates were changed.Into new plates, 400 µL of 10 mM H 2 O 2 was introduced after 72 h; this time point was then taken as 0 h and the number of dead C. elegans was noted every hour to calculate the survival rate using criteria similar to the lifespan assay.The experiment was repeated three times, as shown in Table S7.

Chronic Oxidative Stress Resistance Assay
At least 60 synchronized L4-stage C. elegans were selected and transferred to DnAEcontaining or DnAE-free NGM culture dishes.Then, 200 µL of 10 mM glyphosate solution was added to each culture dish.The C. elegans were relocated to new plates and counted for dead and surviving numbers every 48 h until all the worms were dead.The assay was carried out in three parallel groups.The experiment was repeated three times, as shown in Table S7.

Thermal Stress Resistance Assay
The dosing and control groups were treated with or without 1 mg/mL DnAE.After transferring at least 40 synchronized L4-stage nematodes into NGM dishes, they were treated with either 1 mg/mL DnAE or solvent only and cultivated for 5 days at 20 • C.Then, the temperature was raised to 37 • C and the number of dead worms was counted every hour.When the worms no longer responded to being touched by the platinum wire, they were considered dead.The experiment was repeated three times, as shown in Table S7.

Determination of Lipofuscin
Wild-type nematodes were cultured according to the lifespan assay.After 5 and 10 days of culture, the fluorescence intensity was observed using an inverted fluorescence microscope (NIKON Eclipse Ts2R, Nikon, Tokyo, Japan) under conditions of excitation at 340 nm and emission at 430 nm.The average fluorescence intensity of the entire body of C. elegans was then analyzed using ImageJ v1.53.The experiment was repeated three times and at least 20 nematodes were included in each group, as shown in Table S6.

Determination of ROS Levels
ROS levels were quantified using the cell membrane-permeable reactive oxygen species (ROS) detection probe H2DCFH-DA.After synchronization of the wild-type nematodes, L4-stage nematodes were selected and transferred to new NGM dishes with or without 1 mg/mL DnAE every 48 h.In the positive control group, an additional 4 mM paraquat was added.After 5 days of incubation, the nematodes were transferred to an M9 buffer containing 100 µM H2DCFH-DA and incubated for 2 h at 20 • C in a light-protected incubator.The fluorescence intensity was observed with an inverted fluorescence microscope (Nikon eclipse Ts2R) at an excitation wavelength of 488 nm, and the average fluorescence intensity of the entire body was subsequently analyzed using ImageJ.At least 20 C. elegans were used per group and the experiment was repeated three times, as shown in Table S8.

Determination of SOD Activity
This experiment involved a treatment group and a control group treated with or without 1 mg/mL DnAE.After synchronization at the L4 stage, approximately 1000 nematodes were transferred to each group every 48 h.After 5 days of normal incubation, the nematode lysate was prepared using an ultrasonic cell disrupter under the following conditions: ultrasonic power of 200 W, cycle time of 20 s, interval of 10 s, and 10 cycles.The total protein concentrations of the lysate were quantified using the BCA protein assay kit (Beyotime, P0012).SOD activity was detected using a commercial kit (Nanjing Jiancheng Bioengineering Institute, A001-3), mostly following Chen Xu's protocol [70].Briefly, the lysate was added to a 96-well plate supplemented with superoxide anion radical and 2-(4iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt (WST-1) and incubated at 37 • C for 20 min to produce SWT-1 formazan.The absorbance of the formazan was measured at 450 nm under a microtiter plate reader (Thermo, Varioskan Flash, Waltham, MA, USA), which was used for calculating the activity of SOD following the manufacturer's instructions.The experiment was repeated three times, as shown in Table S9.

Quantitative Analysis of Fluorescence Intensity
Through fluorescence intensity quantification analysis, we used the mutant strains BZ555, NL5901, CL2166, CF1553, and TJ375 to analyze whether DnAE affects dopaminerelated neuronal loss, the aggregation of α-synuclein, and the expression of GST-4, SOD-3, and HSP-16.2.Each plate contained 50 µM FUDR to inhibit egg hatching.The BZ555 mutant strain was subjected to a one-hour immersion in a 50 mM solution of 6-hydroxydopamine (6-OHDA) to serve as the model group for dopamine neuron loss.In contrast, the positive control group was treated with 2 mM levodopa (L-DOPA).The TJ356 mutant strain was subjected to a one-hour treatment at 35 • C before imaging as a stress model.The establishment of the CL2166 and CF1553 model groups followed the same strategy described in Section 5.11.The DR model was established with the NL5901 strain, consistent with previous reports [40].Adult worms were transferred to NGM plates without OP50 every two days and cultured for five days.Approximately 60 late L4 larvae or young adults (mutants) were transferred to plates containing 1 mg/mL DnAE or etOH and cultured with inactivated OP50 at 20 • C until day 5.An inverted fluorescence microscope (Nikon eclipse Ts2R) was used to take photographs.ImageJ was used to analyze the average fluorescence intensity in the heads of the BZ555 and CF1553 mutants, while the overall average fluorescence intensity was analyzed for the remaining mutant strains.Real-time images were obtained from at least 20 worms per group and the experiment was repeated three times, as shown in Tables S10 and S12.

Nuclear Localization of DAF-16
As described for the lifespan assay, the TJ356 strain was cultured at 20 • C for four days.Subsequently, it was treated with freshly prepared 4 mM paraquat for 24 h [71].The nuclear translocation of DAF-16 was observed under a fluorescence inverted microscope and images were taken.The obtained data, which were labeled uniformly, were presented to a third party.The data were categorized into three types: nuclear entry, intermediate state, and cytoplasmic entry.The quantity of each category was counted and the corresponding proportions were calculated.The experiment was repeated three times and the number of C. elegans per treatment was not less than 20, as shown in Table S10.

Paralysis Assay
The CL4176 worms were incubated at 15 • C until the L3 stage, transferred to NGM plates containing 1 mg/mL DnAE, and incubated at 25 • C, according to the methods of Drake, Jennifer [42].Paralyzed nematodes were counted every 2 h.The DR model group underwent the same treatment as described in Section 5.13 for NL5901.This experiment was independently repeated three times with a minimum of 40 worms in each group, as shown in Table S13.

Quantitative Real-Time PCR Assay
Approximately 2000 synchronized young adult nematodes were transferred to DnAEcontaining or DnAE-free NGM plates and then maintained at 20 • C for 24 h.Total RNA was extracted using RNAiso Plus (Takara, Kyoto, Japan) and converted into cDNA with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA).Quantitative real-time PCR was carried out using Power SYBR Green PCR Master Mix (Applied Biosystems, Waltham, MA, USA) in a QuantStudio 6 Flex system.The expression levels of the genes were calculated using the 2 −∆∆CT method and normalized to the expression of the cdc-42 gene.The statistical significance of the differences was determined using the t test.The experiment was repeated three times, as shown in Tables S14 and S15.

Statistical Analysis
All the experiments were performed in triplicate.The data are presented as the mean ± SEM unless specifically indicated otherwise.All the statistical analyses included t tests or log-rank tests.All the figures were generated using GraphPad Prism 9, SPSS 26.

Figure 1 .
Figure 1.Total ion chromatograms of DnAE in positive ion mode (A) and negative ion mode (B).* Confirmed with standards.** Tentatively identified based on library and/or literature data.The bold numbers: indicate the corresponding numbers in Table1.

Figure 2 .
Figure 2. Effect of DnAE on the lifespan and healthspan of wild-type C. elegans (N2).(A) Survival of N2 worms treated with or without different concentrations of DnAE.n ≥ 60. (B) Survival of N2 worms treated with EtOH (214 µmol/mL, consistent with the concentration of alcohol contained in 1 mg/mL DnAE) or ddH2O.n ≥ 60. (C) Average and total number of eggs produced per hermaphroditic worm.n ≥ 20.(D) Number of pharyngeal pumping events by worms.(E) Number of bending events in worms after 20 s. n ≥ 20.(F) Representative fluorescence images show lipofuscin levels in N2 after 5 days.The bar chart indicates the fluorescence intensity of N2 treated with DnAE for 5 and 10 days.(n ≥ 20, *** p < 0.001, ***** p < 0.00001, t test).

Figure 2 .
Figure 2. Effect of DnAE on the lifespan and healthspan of wild-type C. elegans (N2).(A) Survival of N2 worms treated with or without different concentrations of DnAE.n ≥ 60. (B) Survival of N2 worms treated with EtOH (214 µmol/mL, consistent with the concentration of alcohol contained in 1 mg/mL DnAE) or ddH 2 O. n ≥ 60. (C) Average and total number of eggs produced per hermaphroditic worm.n ≥ 20.(D) Number of pharyngeal pumping events by worms.(E) Number of bending events in worms after 20 s. n ≥ 20.(F) Representative fluorescence images show lipofuscin levels in N2 after 5 days.The bar chart indicates the fluorescence intensity of N2 treated with DnAE for 5 and 10 days.(n ≥ 20, *** p < 0.001, ***** p < 0.00001, t test).

Figure 3 .
Figure 3. DnAE increased heat stress tolerance by upregulating the expression of hsf-1 and HSPs.(A) Survival curves of N2 cultured at 37 • C; p was calculated using the log-rank test.n ≥ 60. (B) Effects Molecules 2024, 29, x FOR PEER REVIEW

Figure 8 .
Figure 8. DnAE increases stress tolerance, delays the progression of aging-related diseases, and extends the lifespan of C. elegans via DAF-16 and HSF-1.Red arrow: stimulatory effect, identified by our experiments; black arrow: stimulatory effect; dash line arrow: speculated stimulatory effect; dash line: translocation process; solid "T" line: inhibitory effect.

Figure 8 .
Figure 8. DnAE increases stress tolerance, delays the progression of aging-related diseases, and extends the lifespan of C. elegans via DAF-16 and HSF-1.Red arrow: stimulatory effect, identified by our experiments; black arrow: stimulatory effect; dash line arrow: speculated stimulatory effect; dash line: translocation process; solid "T" line: inhibitory effect.
* Confirmed with standards.** Tentatively identified based on library and/or literature data.C2: